In an aqueous environment, peptides and proteins fold into conformationally limited structures that have the ability to interact with other molecular species and initiate biological responses. These interactions can be defined as, but are not limited to, protein-protein, ligand-receptor, substrate-enzyme, antigen-antibody and protein-nucleic acid recognition. Disruption or enhancement of these macromolecular interactions often cannot be accomplished with small molecules. Using larger molecules to disrupt or enhance biological interactions requires the mimicking of the protein's native spatial configuration. Using a large molecule that has a close spatial homology to the native protein ensures high affinity targeting of the desired species. Compounds that can specifically disrupt or enhance these macromolecular interactions would be invaluable tools in bionanotechnology, and, potentially, as lead compounds for the development of new drugs.
The conformation of a protein is defined by secondary structural elements, which include beta-sheets, beta-turns, alpha-helices, 310-helices, pi-helices, and loops. These secondary structures participate broadly in moderating biological processes including specific recognition of macromolecular interactions. Exposed secondary structural elements on the surfaces of proteins are often important for the specific recognition of other biomolecules. Short peptides, having less then 20 amino acids, corresponding to the secondary structural region in a protein do not remain in the same secondary structure once the peptide is excised from the protein. Short peptides, of less then 20 amino acids, that can adopt secondary structural elements are expected to be useful models for the predictable design of bioactive molecules.
Many peptides are highly flexible and do not fold into unique conformations; thus these peptides cannot maintain the structure that they adopt when they are part of a full-length protein. This flexibility decreases the affinity of the peptide for a macromolecular target because some of the free energy of binding is squandered in paying the entropic cost of constraining the peptide into the proper conformation for binding. The conformational flexibility of peptides thus significantly compromises their potential use as drugs. Many techniques have been developed to constrain the peptides into singular conformations (hydrogen-bond surrogate, disulfide bridges, and other side chain cross linking methods). Constrained peptides having high binding affinities to macromolecular target have found wide use in analyzing structure-function relationships within macromolecular interactions.
Constrained peptide scaffolds, capable of presenting a sequence of interest as a conformationally-restricted body have been identified, including thioether-linked cyclic structures (Souers et al. (1999) JACS 121 1817-1825), hydrogen-bond surrogates (Wang et al. (2005) Angew. Chem. Int. Ed. 44, 6525-6529, U.S. Pat. No. 7,202,332), leucine-zipper motifs (Martin et al. (1994) EMBO J. 13:5303-5309), tryptophan-zipper scaffold that forms stable beta-hairpins in solution (U.S. Pat. No. 6,914,123) and many others. Beta turns are commonly involved in the molecular recognition process and are thus desirable for disrupting or enhancing binding (Smith & Pease (1980) CRC Crit. Rev Biochem 8:315-399). Currently, a commercial peptide drug, based on a beta-turn, has been approved by the FDA for clinical use: Octreotide (brand name Sandostatin®, Novartis Pharmaceuticals). Many of the identified beta-turn peptides are cyclopeptides that are synthesized by covalently attaching the N and C termini of the peptide (Suat Kee et al. (2003) Current Pharm. Design 9, 1209-1224). Many of these cyclic peptides still have a large degree of conformational heterogeneity and need to be further stabilized.
Peptides composed of hydrophobic, alternating D and L-amino acids (D,L-peptides) fold into beta helices in nonpolar environments. A given D,L-peptide usually forms a mixture of beta-helical species, including single-stranded, parallel double-stranded, and antiparallel double-stranded beta helices. One class of peptide sequences that display such conformational promiscuity is oligo D,L-valine peptides (Lorenzi et al. (1982) JACS 104, 1728-1733). To reduce the conformational heterogeneity of the beta-helices, Clark et al. developed a strategy for limiting the number of structural states available to the valine beta helix in nonpolar organic solvents (Clark et al. (2006) JACS 128, 10650-10651). We note that this disclosure was limited to the design of peptides that fold into the correct structures only in nonpolar organic solvents; however, many potential applications of constrained peptides—including the design of lead compounds for the development of new drugs—require that the peptide fold into the correct structure in aqueous media. Satisfying this requirement can be difficult because, in aqueous media, water competes for hydrogen bonding partners with the internal hydrogen bonds that stabilize the peptide's conformation, thus disrupting the peptide's structure. Thus, the design of beta-helical peptides that have well-defined and predictable structures in aqueous media would pave the way to a host of potential applications in biotechnology.
This beta-helical structure use as a scaffold for displaying libraries of peptide/peptidometric side chains as a way of disrupting or enhancing macromolecular interactions. Previously, nearly all beta-helical peptides examined were only soluble in organic solvents, and none of this work suggested that the correct conformation could be attained in aqueous media. Alexopoulos et al. (2004) Acta Cryst. D60, 1971-1980, reported a water-soluble D,L-peptide designed to fold into a beta-helical structure. Using x-ray crystallography, the authors showed that the peptide, an oligo D,L-tyrosine, forms a beta-helical dimer in the solid state. Only indirect evidence for the aggregation of the peptide in aqueous media was provided. The aggregation was not demonstrated to be due to the formation of a beta-helical dimer.